META-ANALYSIS OF ENDOPHTHALMITIS AFTER INTRAVITREAL INJECTION OF ANTI–VASCULAR ENDOTHELIAL GROWTH FACTOR AGENTS: Causative Organisms and Possible Prevention Strategies

Purpose: To report the rates of endophthalmitis and the spectrum of causative organisms after intravitreal injection of anti-vascular endothelial growth factor agents and possible prevention strategies. Methods: Meta-analysis of the U.S. literature from 2005 to 2009 reporting endophthalmitis bacterial isolates after intravitreal injection of anti-vascular endothelial growth factor agents and comparison with reports of endophthalmitis bacterial isolates after intraocular surgery in the United States. Results: Endophthalmitis after intravitreal injection occurred in 52 of 105,536 injections (0.049%) (95% confidence interval [CI], 0.038-0.065%). Among 50 cases of endophthalmitis with bacterial culture isolates, 24 (48.0% [95% CI, 34.8-61.5%]) were culture negative and 26 (52% [95% CI, 38.5-65.2%]) were culture positive. Among the 26 culture-positive isolates, causative organisms were coagulase-negative Staphylococcus in 17 cases (65.4% [95% CI, 46.0-80.6%]), Streptococcus species in 8 cases (30.8% [95% CI, 16.5-50.2%]), and Bacillus cereus in 1 case (3.8% [95% CI, 0.9-19.0%]). Streptococcus species were significantly more frequent after intravitreal injection than after intraocular surgery in the Endophthalmitis Vitrectomy Study (29 of 226 isolates, 9.0% [95% CI, 6.3-12.6%], P = 0.005), a report on clear corneal cataract surgery endophthalmitis (6 of 73 isolates, 8.2% [95% CI, 3.9-16.8%], P = 0.022), and a report on postvitrectomy endophthalmitis with no cases of Streptococcus species. Conclusion: Streptococcal isolates are approximately three times more frequent after intravitreal anti-vascular endothelial growth factor injection than after intraocular surgery. Strategies to consider minimizing oropharyngeal droplet transmission may include avoiding talking, coughing, and sneezing or wearing surgical masks.

E ndophthalmitis after penetrating ocular procedures is a rare but devastating complication. The range of acute postoperative endophthalmitis in the United States has been reported to be 0.04% to 0.076%. [1][2][3][4] The most common causative organisms are coagulase-negative Staphylococcus species.
Endophthalmitis after intravitreal injection of antivascular endothelial growth factor (VEGF) agents (pegaptanib, bevacizumab, and ranibizumab) is similarly rare. Reported endophthalmitis rates vary from 0.019% 5 to 0.54%. 6 These rates are low despite the injections being performed in the office setting. To date, there have been no reports on the spectrum of causative organisms observed after endophthalmitis after anti-VEGF agent injection compared with those observed in acute postoperative endophthalmitis. This study describes a metaanalysis performed to determine the spectrum of causative organisms in endophthalmitis after intravitreal injection of anti-VEGF agents compared with those in the setting of acute postoperative endophthalmitis.

Materials and Methods
Using the National Library of Medicine PubMed interface (www.pubmed.gov), a literature search was performed. All articles containing the keywords ''endophthalmitis'' and ''injection'' during the years 2005 to March 22, 2010, inclusive (Medline search date: March 22, 2010) were identified. Additionally, the articles were reviewed for references that might identify additional relevant studies, and these were also reviewed. Those articles in which the title suggested that the publication might contain information on anti-VEGF agent injection endophthalmitis were reviewed to assess whether the article met inclusion criteria. The inclusion criteria were threefold. First, articles were required to report on endophthalmitis after intravitreal injection of an anti-VEGF agent. Second, articles were required to provide the endophthalmitis culture results, including bacterial species that were isolated (''isolates''). Third, articles were required to report on patient populations in the United States, given the possible variation in procedures, settings, and bacterial colonization patterns elsewhere. Articles that fulfilled all inclusion criteria were included in the meta-analysis. Additionally, references in the selected articles and those of review articles on endophthalmitis were used to identify additional publications that might be eligible for inclusion. Any additional publications that met the inclusion criteria were also included in the meta-analysis. Articles that reported on endophthalmitis rates but did not include the causative organism information or those that had no cases of endophthalmitis were excluded. A large series of endophthalmitis after surgery performed in the operating room that provided information on causative organisms, or isolates, from the United States were selected for comparison.
Statistical analyses between the meta-analysis and the comparative postoperative studies were made using the chi-square test. A funnel plot was performed to assess systematic publication bias of the metaanalysis.

Results
The initial PubMed search returned a total of 432 articles. After additional inclusion of referenced articles, there were 24 articles that provided information on endophthalmitis and causative organisms after anti-VEGF agent injection. After all inclusion criteria were applied, there were 16 remaining articles that fulfilled all inclusion criteria. The relevant information from these articles is summarized in Table 1. [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] Studies reporting endophthalmitis and causative organisms after intravitreal injection outside the United States were reports by Aggio et al, 21 Alkuraya et al, 22 Artunay et al, 23 and Yenerel et al, 24 and Wu et al. 25 A report by Lee et al 26 was excluded because the authors convincingly tracked the occurrence of two cases of Serratia marcescens endophthalmitis to pharmacy contamination. In the VEGF Inhibition Study in Ocular Neovascularization (VISION) trial 27,28 publications, causative organisms of the 12 cases of endophthalmitis were not reported, and thus, these cases were not considered in the meta-analysis.
A total of 54 cases of endophthalmitis were considered in the meta-analysis. The cumulative rate of endophthalmitis was determined to be 52 cases after 105,536 injections (0.049%) (95% confidence interval [CI], 0.038-0.065%). Two case reports were not included in the rate calculation because no denominator was provided in the publications for these cases. A culture was obtained for 50 cases, 24 ( In comparison, the causative organisms and their relative rates among the 3 largest studies of postoperative endophthalmitis with causative organisms after surgery in the operating room are summarized in Table 2. [29][30][31] For comparison, isolates of normal conjunctival flora from patients about to undergo intravitreal injection for the first time are summarized in Table 3. 32 Comparing the distribution of organisms in the meta-analysis with these postoperative reference studies, Streptococcus species were significantly more frequent after intravitreal injection than after intraocular surgery in the Endophthalmitis Vitrectomy Study 29,33 (29 of 323 isolates, 9.0%, P = 0.005), a recent report on post-clear corneal cataract surgery and a report on postvitrectomy endophthalmitis 31 with no cases of Streptococcus species. The report on normal conjunctival flora preinjection also indicated fewer Streptococcus species (9 of 129 isolates, 7%, P = 0.0016). A funnel plot estimating endophthalmitis rates against the number of injections reported in each study is shown in Figure 1. 34 Among 4 small studies with the number of injections ,2,000, there was a wide spread of overestimates of endophthalmitis rates, ranging from 0.24% to 0.55%. Among large studies with the number of injections ranging from 3,125 to 30,746, the estimated rate of endophthalmitis ranged from 0.02% to 0.1%. Therefore, there may be ''small study effects'' overestimating the endophthalmitis rate, and it is unlikely that a systematic publication bias exists for reported rates of endophthalmitis after intravitreal anti-VEGF injections.

Discussion
This meta-analysis of endophthalmitis after intravitreal anti-VEGF injections found a significantly greater rate of endophthalmitis cases caused by Streptococcus species than has been reported for postoperative endophthalmitis. Streptococcus species, which comprise at least 41% culturable adult salivary flora, 35,36 are thought to contaminate operative fields by aerosolization or droplet spread [37][38][39][40][41] and may be related to the difference in causative organisms in these 2 settings.
Staphylococcal organisms (coagulase-negative Staphylococcus, Staphylococcus aureus, and methicillin-resistant S. aureus) occur rather uniformly at rates of approximately 70% to 80% across all studies (Tables 1-3), including the current meta-analysis of anti-VEGF injections (Table 1). However, the rate of Streptococcus species as a causative organism of postoperative endophthalmitis or preexisting colonization of the conjunctiva is in the range of 0% to 9% (Tables 2 and 3), which contrasts dramatically with the 30.2% rate of streptococcal endophthalmitis after intravitreal injection (Table 1) found in the meta-analysis. This three to four times higher rate of endophthalmitis from Streptococcus species suggests that the spectrum of organisms is different than in the operating room setting and that Streptococcus species are more common causatives of endophthalmitis after intravitreal injection.
A review of the ophthalmic literature indicates no explanation for the different streptococcal endophthalmitis rates after intravitreal anti-VEGF agents compared with the postoperative setting. However, there is compelling evidence that aerosol contamination of the surgical field by respiratory flora may be contributory. Dural puncture (i.e., lumbar puncture for spinal anesthesia) is a procedure similar to intravitreal injection in that it involves needle penetration into a nutrient-rich body cavity in a nonoperating room setting. The rate of postdural puncture meningitis is low (0.2-1.8/10,000) 42-44 compared with that of postintravitreal injection endophthalmitis (4.9/10,000 in this meta-analysis).
A closer look at the factors that have been determined to increase the risk of postdural puncture  45 in similar studies investigated whether wearing soft pleated face masks affects agar dish contamination. These reports determined that the absence of a surgical mask was associated with a statistically significant increase in colonies per exposed plate. They recovered a-hemolytic Streptococci, nonhemolytic Streptococci, and coagulase-negative Staphylococci. 40 O'Kelly and Marsh 39 and Phillips et al 45 in similar studies investigated the effect of talking and wearing a face mask on agar plate contamination to assess procedure infection risk. In these studies, there was a significant increase in colony counts when not wearing a mask and talking compared with not talking or wearing a mask. Furthermore, O'Kelly and Marsh 39 suggest that not talking is nearly as effective as wearing a face mask in preventing agar plate contamination and thus reducing procedure infection risk from respiratory tract flora.
Another procedure that is similar to an intravitreal injection is intraarticular injection. A recent report by Reeves and Hovart 46 implicates the absence of a face mask as the likely cause of infection with a-hemolytic Streptococcus after an intraarticular knee injection. In this report, infection risk in a retrospective series of injections is associated with talking when no mask was worn. 46 There is also direct evidence that respiratory droplet transmission from the health care provider may be the source of procedure-related infection. Using molecular techniques, causative organism of meningitis cases after dural puncture procedures was indistinguishable from those recovered from the procedurist's mouth. 37,41,47 Sheretz et al 38 describe an outbreak of 5 cases of methicillin-resistant S. aureus infections, 5 cases of pneumonia, 1 case of bacteremia, and an additional 3 cases of colonization with methicillinresistant S. aureus over a 3-week period. Investigation traced the probable source of the outbreak to a physician colonized with the same organism, and that during the outbreak, he had a viral upper respiratory infection (URI). Further investigation also showed that while this individual had very little ''bacterial shedding'' when not experiencing a viral URI, he did shed a large amount of bacteria when purposely reinfected with a rhinovirus (Type 39). This observation suggests that viral URI increases bacterial shedding. A surgical mask was able to minimize dispersal of bacteria during the study of the URI episode. 38 These publications lend critical evidence that supports droplet or aerosol transmission of infections by aerosolized bacteria from the oropharyngeal tract. Endophthalmitis is usually thought to originate from organisms of the patients' flora. 48 The causative organisms of endophthalmitis among several postoperative studies and a study of conjunctival isolates support the assumption that organisms from the patients' flora caused endophthalmitis (Tables 2 and  3). However, the same pattern does not hold for the causative organisms after intravitreal injection of anti-VEGF agents where this meta-analysis found a substantially greater percentage of Streptococcus species. The limitations of this study include the retrospective nature of the data considered in the meta-analysis, which may introduce bias. It is possible that not all cases of endophthalmitis were reported. In addition, meta-analyses are inherently subject to publication bias resulting from the possible lack of publications with negative findings (no reports of endophthalmitis) or possible lack of a broader experience because of limitations on total number of studies published on any one topic. Therefore, the rate of endophthalmitis and/ or causative organism may be less accurate. Furthermore, the speculation that aerosolized droplet contamination may be in part responsible for the relatively high rate of streptococcal endophthalmitis is supported by the literature on postdural puncture and intraarticular injections because no direct evidence from the ophthalmic literature exists.
There is valuable insight to be gained from knowledge reported in the nonophthalmic literature regarding droplet transmission as a possible source of some cases of endophthalmitis, [37][38][39][40][41]46 particularly that caused by potentially devastating Streptococcus species. Droplet or aerosol transmission may be enhanced by talking, coughing, or sneezing, and bacterial shedding may be exacerbated by URI. 38 Avoiding talking or wearing a face mask to minimize infection risk with intravitreal injections must be considered to mitigate the risk of droplet or aerosol transmission. Although published consensus statements on proper technique for intravitreal injections have not included the recommendation to wear a face Isolates in a recent study of conjunctival isolates in a patient population about to undergo intravitreal injection in the United States. mask, 49,50 the growing experience with cases of endophthalmitis and the presented evidence for oral flora as causative organisms for streptococcal endophthalmitis provide a strong case for wearing masks or at least eliminating talking during intravitreal injections. The Centers for Disease Control and Prevention's Healthcare Infection Control Practices Advisory Committee issued recommendations in 2005 that for dural puncture procedures, a face mask should be worn. 51 In summary, this meta-analysis suggests that upper respiratory tract organisms are causative of endophthalmitis at a three to four times greater rate after intravitreal injection than after postcataract surgery or postvitrectomy cases of endophthalmitis. Thus, it seems likely that in at least some cases, the transmission of the organisms occurs by aerosolized upper respiratory tract organisms that contaminate the sterile injection field or the injection needle. Those performing intraocular injections should consider taking precautions against droplet contamination, such as not talking, talking with the mouth turned away from the field, or wearing a mask, especially if experiencing an URI, during intravitreal injection procedures. Such measures may help further reduce the already low rate of endophthalmitis after intravitreal injection. Further research to clarify the role of saliva droplet contamination in causing streptococcal endophthalmitis is warranted.